Download Lactate dehydrogenase is crucial for tumor associated macrophage

Lactate dehydrogenase is crucial for tumor associated macrophage
protection of multiple myeloma cells against chemotherapy
Carolyn Stierhoff, Enguang Bi, Lintao Liu, Yong Lu, Xingzhe Ma, Jianfei Qian, Qiang Wang, Gang Xue, Maojie Yang, Tianshu Li, Qing Yi
CLEVELAND CLINIC LERNER RESEARCH INSTITUTE
ABSTRACT
Multiple myeloma (MM) is a cancer characterized by an
accumulation of malignant plasma cells in the bone marrow.
Although chemotherapy is the most effective treatment, the
majority of patients experience relapse. The major cause of
treatment failure is the development of multidrug resistance. Thus,
overcoming drug resistance will greatly improve patient survival.
The enzyme lactate dehydrogenase (LDH) plays an important role
in anaerobic respiration by converting pyruvate into lactate in the
absence or low supply of oxygen. It has been observed that tumorassociated macrophages (TAMs) present in tumor environments
can protect MM from chemotherapy treatment. We discovered that
an LDH knockdown in myeloma cells decreased TAMs’ role in
cell protection against chemotherapy treatment. Also, we found
that, without LDH, macrophages were not able to differentiate into
TAMs successfully. These results suggest that LDH activity in
tumor environments promotes tumor survival, stimulates
tumorigenesis, and manipulates local macrophages into
performing pro-tumor functions.
Methods
• RT-PCR and western blotting to determine the efficiency of the LDH
knockdown.
• Flow cytometry with an apoptotic signaling assay to determine
knockdown effects in myeloma/macrophage co-cultures.
• RT-PCR to identify the expression patterns of known TAM markers
with an LDH knockdown.
• Flow Cytometry with a cell viability assay to determine TAM
proliferation with an LDH knockdown.
Figure 3. RT-PCR showing the relative expression of the TAM
markers ARG1 and VEGF. With LDH knockdown, ARG1 and
VEGF expression is reduced.
RESULTS
INTRODUCTION
The standard treatment for MM is chemotherapy with combinations
of drugs such as melphalan, thalidomide, bortezomib, and
dexamethasone1. Some cancer cells and tumors remain resistant or
develop resistance to these treatments. TAMs have been shown to
have both pro- and anti-tumor effects. Some of the anti-tumor
effects include tumor angiogenesis, growth, development, and
2
treatment resistance . In previous studies, it has been shown that, by
affecting apoptotic signaling, TAMs protect myeloma cells with
3
which they are in direct contact .
Figure 4. Flow cytometry showing a decrease in tumorassociated macrophage proliferation with the LDH knockdown.
Figure 1. RT-PCR and western blotting showing that an LDH
knockdown decreases LDH mRNA expression and detectable protein
levels in myeloma cells.
The goal of this study was to explore possible mechanisms for
myeloma cell protection by means of TAMs and their infiltration
into myeloma tumor beds. Previously, other studies have associated
high LDH levels with low prognosis and shortened survival rates for
multiple myeloma patients4. In order to explore the role of LDH in
TAM-associated protection, a gene knockdown of LDH was
performed. A knockdown involves a post-transcriptional silencing of
a gene, in this case LDH, by the use of small RNA molecules which
target gene transcripts for degradation, thus causing reduced
expression of the target gene. In order to aid in development of
more effective chemotherapeutic agents to combat MM, our study
looks to identify the role of LDH in TAMs' protection of myeloma
Figure 2. Flow cytometry using co-cultures of macrophage and myeloma
cancer cells through this knockdown method.
cells representing increased myeloma cell death with melphalan treatment
and LDH knockdown.
CONCLUSIONS
• An LDH knockdown decreases macrophages’ ability to
protect myeloma cells from melphalan treatment.
• LDH knockdown decreases the expression of known tumorassociated macrophage markers.
• Knocking down LDH in myeloma prevents tumor-associated
macrophages from growing and proliferating.
REFERENCES
1 Multiple Myeloma. American Cancer Society, Jan 2015. Web. 8 March 8, 2016.
http://www.cancer.org/cancer/multiplemyeloma/detailedguide/multiple-myeloma-treating-chemotherapy.
2 Quatromoni, Jon G, and Evgeniy Eruslanov. “Tumor-Associated Macrophages: Function, Phenotype, and Link
to Prognosis in Human Lung Cancer.” American Journal of Translational Research 4.4 (2012): 376–389. Print.
3 Zheng, Yuhuan et al. “Macrophages Are an Abundant Component of Myeloma Microenvironment and Protect
Myeloma Cells from Chemotherapy Drug–induced Apoptosis.” Blood 114.17 (2009): 3625–3628. PMC. Web. 8
Mar. 2016.
4 Evangelos Terpos et al. “High serum lactate dehydrogenase adds prognostic value to the international myeloma
staging system even in the era of novel agents.” Eur J Haematol. 2010;85:114–119. PMC. Web. 8 March 2016.
ACKNOWLEDGEMENTS
Thank you to the Yi Lab and Cleveland Clinic Lerner Research Institute